Fig 1: Phagocytosis assay for WT, CRT−/−, and ERp57−/− cells. CellTracker-labeled Jurkat WT, CRT−/−, or ERp57−/− cells were untreated or treated with oxaliplatin, followed by co-incubation with F4/80-labeled primary mouse macrophages. (A) Representative flow cytometry plots showing input target (Jurkat) and/or effector macrophages. The dual positive gated events are indicative of phagocytosis. (B) The calculated phagocytosis index depicted as bar graphs for the indicated target cells and treatments. Plotted are the mean ± S.D.; n = 3; p-values: *** < 0.001; ** < 0.01; ns, not significant. Data shown are representative of at least three independently conducted experiments.
Fig 2: Depletion of ERp57 increases apoptosis in hypoxia. (A) Annexin/ propidium iodide staining was performed to detect early and progressed apoptosis after radiation with 10 Gy or ERp57 KD. Annexin was conjugated to Pacific Blue and thereby designated as „Pacific Blue-H “ in the charts. Propidium iodide as indicator for progressed apoptosis was designated as PE-H. The diagram shows one representative result out of three performed experiments. The induction of apoptosis due to the respective treatment was illustrated as n-fold induction to the untreated sample. Early and late apoptosis were summed as comprehensive apoptosis. (B) Caspase-3 activity was measured 96 h after KD and 48 h after radiation with 10 Gy. Cells were in hypoxia for 72 h. (C) After the same treatment as in (B) pro-apoptotic proteins p53 and PUMA were checked by Western blot.
Fig 3: qRT‐PCR analysis of ER stress related genes. The mean fold‐increase of (a) BiP, (b) PDI and (c) bZIP60 are indicated following the co‐expression of human calreticulin with HIV gp140. The 18S rRNA was used for the normalization of cDNA amount. A control, comprising of plants infiltrated with A. tumefaciens, transformed with the empty pEAQ‐HT vector, was included for comparison. Error bars represent standard deviation of three biological replicates. (*; P < 0.05, **; P < 0.01, ***; P < 0.001, NS = not significant). (empty vector = infiltration with A. tumefaciens transformed with pEAQ‐HT, CRT = CRT expression only, gp140 = gp140 expression only, CRT + gp140 = co‐expression of gp140 and CRT).
Fig 4: Analysis of protein–protein interaction for ERp57 with PDPK1, AKT, PLK1 and c-Myc. Plasmid pe-N1-ERp57-V5 was co-transfected with vectors CMV-3xFlag-hPDPK1, pCDNA3-HA-AKT1, pEYFP-PLK1 or pCMV4a-Flag-c-Myc into HEK293T cells. For all IP experiments, ERp57 was pulled with an anti-V5 antibody (V5-IP) and complex formation with PDPK1, AKT, PLK1 and c-Myc was determined by western blot analysis using anti-FLAG, anti-HA, anti-GFP and anti-c-Myc antibodies, respectively. 1–3% of total cell lysate was loaded as input.
Fig 5: Treatment of various T-ALL lymphoblasts with 9EG7 and oxaliplatin reduces surface exposure of CRT and ERp57. Flow gMFI plots for surface CRT (A) and surface ERp57 (B) for T-lymphoblasts treated with combinations of oxaliplatin and/or 9EG7 integrin antibodies. DND-41, THP-6, REX, and SUP-T1 are T-ALL cell lines. T048-D and T048-R are PDX T-ALL samples from a single patient taken at diagnostic and relapsed disease. Plots are the mean ± S.D.; n = 3; p-values: *** < 0.001; ** < 0.01; * < 0.05. Data shown are representative of at least two independently conducted experiments. (C) Immunoblot assay for expression of CRT, ERp57, and GAPDH in lysates of the indicated cells.
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